The job of our immune system is to recognize foreign invaders and to eliminate the threat while sparing self-tissues. Such discrimination occurs at the level of T cell receptor (TCR) recognition of peptide/MHC molecular complexes on target cell surfaces. Signals arising from recognition of self-peptide/MHC molecules (self-ligands) by developing thymocytes determine lineage commitment and selection of the TCR repertoire. However, the role of self-ligand interaction in peripheral mature T cells is controversial. Specifically, how the presence of self-ligands influences lymphocyte function and migratory behavior in vivo remains unclear. Until recently, such determination largely comes from indirect experimental evidence. With the aid of 2-photon laser scanning microscopy, however, investigators can now begin to understand intercellular dynamics within an intact tissue microenvironment such as the lymph node. An example of the utility of this powerful imaging tool is the uncovering of the role inflammatory chemokines play in the orchestration of multi-cellular cooperation during the intranodal induction of a primary immune response8. In the current study, we propose to provide direct, high-resolution data addressing the impact of self-ligand surveillance by circulating mature lymphocytes in the lymph nodes. We also plan to undertake a series of studies aimed to delineate the relative contribution of various self-ligand-expressing cells during immune surveillance by naive lymphocytes as well as other specialized cells such as regulatory T cells. This will be accomplished by utilizing the state-of-the-art 2-photon laser scanning microscopy available in our own laboratory, combined with classical immunological techniques and mouse bone marrow chimera manipulations. Success in this endeavor will provide new insights into how self-ligands contribute to the global lymphocyte behavior and function in vivo, and will set the stage for a detailed understanding of how self-peptide/MHC interactions shape the responses of the T cell populations against cognate antigens encountered during infection and vaccination. A detailed understanding of the intricate and complex cellular communication in the tissue microenvironment will allow future inquiries into the in vivo cellular mechanisms that lead to the development of dysregulated state of self-tolerance, such as autoimmunity, cancer and chronic infection. PUBLIC HEALTH RELEVANCE: Recognition of positive-selecting self-ligands plays a crucial role in developing T lymphocytes during thymic education. However, the role of such self-peptide antigen recognition in the periphery for mature T lymphocytes is controversial. The current study seeks to investigate the role self-peptide antigens play in the in vivo behavior of T lymphocytes in secondary lymphoid organs. We aim to provide high-resolution data addressing the impact of self-ligand interactions, and to seek the relative contribution of various stromal and immune cellular subsets in affecting the migratory behavior of naive and regulatory T lymphocytes in vivo. The proposed studies will set the stage for proper understanding of how self-peptide/MHC interactions shape the responses of these T cell populations against cognate antigens seen during infection and vaccination. Successful execution of this project will also provide new insights into potential mechanisms underlying the development of autoimmunity, induction of tumor tolerance and maintenance of homeostatic immune responses.